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X chromosome inactivation in human developmentCatherine Patrat1,2,*, Jean-François Ouimette3 and Claire Rougeulle3,*

ABSTRACTX chromosome inactivation (XCI) is a key developmental processtaking place in female mammals to compensate for the imbalance inthe dosage of X-chromosomal genes between sexes. It is aformidable example of concerted gene regulation and a paradigmfor epigenetic processes. Although XCI has been substantiallydeciphered in the mouse model, how this process is initiated inhumans has long remained unexplored. However, recent advances inthe experimental capacity to access human embryonic-derivedmaterial and in the laws governing ethical considerations of humanembryonic research have allowed us to enlighten this black box. Here,we will summarize the current knowledge of human XCI, mainlybased on the analyses of embryos derived from in vitro fertilizationand of pluripotent stem cells, and highlight any unansweredquestions.

KEY WORDS: X chromosome inactivation, Human, Germline,Embryo, XIST, XACT, Pluripotent stem cells

IntroductionThe formation of heteromorphic (see Glossary, Box 1) sexchromosomes during the evolution of mammals has created adosage imbalance for sex-linked genes between sexes. Althoughthis imbalance is well tolerated for the male-specific Ychromosome, probably because of its highly specialized and lowgene content, this is not the case for the X chromosome, whichcovers ∼1.5 Mb of DNA and harbors up to a thousand genes, manyof which serve fundamental cellular functions. In metatherians andeutherians, the increased dose of X-linked genes in females iscompensated for by the functional exclusion of one of the two Xchromosomes, a process referred to as X chromosome inactivation(XCI; see Glossary, Box 1). During XCI, several hundreds ofphysically linked loci are concomitantly and stably silenced (RobertFinestra and Gribnau, 2017). Of note, some genes are refractory toXCI and escape this process, but the proportion of such genes variesfrom one species to another (Carrel and Brown, 2017).Mice have long been the leading model for X-inactivation studies

in placental mammals, and have thus served to elucidate thedevelopmental regulation of this process and to identify most of themolecular mechanisms and factors involved. XCI is tightlyregulated, established early during embryonic development andthen stably maintained for the entire in utero and adult life. In thegermline, however, reactivation of the X chromosome occursconcomitantly to global epigenetic reprogramming during

primordial germ cell (PGC; see Glossary, Box 1) specification(Payer et al., 2011). More subtle exploration revealed two waves ofXCI in mouse (Fig. 1). First, soon after fertilization and zygoticgenome activation, XCI is established in an imprinted manner, withsystematic inactivation of the paternal X chromosome (Fig. 1; Maket al., 2004; Okamoto et al., 2004). This form of X-inactivation ismaintained in the extra-embryonic lineages, but reversed in theinner cell mass of early blastocysts, in which the two Xchromosomes are transiently active (Mak et al., 2004; Okamotoet al., 2004; Takagi, 2003). Subsequently, random XCI (rXCI; seeGlossary, Box 1) is initiated in cells of the embryo proper, and givesrise to individuals in whom the maternal and paternal Xchromosomes are active in roughly half of the cells each (Dupontand Gribnau, 2013).

At the molecular level, XCI is triggered by the long noncodingRNA (lncRNA; see Glossary, Box 1) X-inactive specific transcript(XIST; see Glossary, Box 1), which is expressed from and remainsassociated with one of the twoX chromosomes (Borsani et al., 1991;Brockdorff et al., 1991; Brown et al., 1991). XIST accumulationforms a cloud-like structure in the nucleus, visible using RNAfluorescence in situ hybridization (RNA-FISH), and acts as aplatform for the recruitment of various complexes (Moindrot andBrockdorff, 2016). Through mechanisms that remain unclear, thismodifies the organization of the decorated chromosome at multiplelevels and prevents gene expression. One of the hallmarks of theinactive X chromosome (Xi; see Glossary, Box 1) is itsheterochromatic and condensed nature (Barr and Bertram, 1949).Heterochromatization of the Xi involves a sequential, yet rapid,switch in post-translational modifications of histones, with pan-acetylation and tri-methylation of lysine 4 of histoneH3 (H3K4me3)being removed and replaced by other marks such as tri-methylationof H3 lysine 9 and 27 (H3K9me3 andH3K27me3, respectively), andubiquitination of H2A at lysine 119 (H2AK119Ub) (Zylicz et al.,2019). The Xi is also enriched for specific histone variants, such asmacroH2A, and displays CpG island hypermethylation (Nora andHeard, 2010). All of these events are triggered, directly orindirectly, by the accumulation of XIST RNA, the expression ofwhich depends on trans-acting factors and on elements located inthe vicinity of the XIST gene, in a region called the X-inactivationcenter (XIC, see below). Several of these elements are noncodingthemselves, and act through a variety of mechanisms that are notyet fully deciphered (Furlan and Rougeulle, 2016).

Despite the compulsory nature of XCI, the hierarchy of eventsleading to XCI and the XCI regulatory network, although largelyconserved, display species specificities (Okamoto et al., 2011;Petropoulos et al., 2016; Vallot et al., 2017). In particular, there ismounting evidence that X chromosome activity, whether duringearly pre-implantation development or in the germline, embracesspecific dynamics in humans (Fig. 1), raising questions as to theuniversality of the underlying regulatory network in eutherianmammals. In this Review, we discuss recent advances andhighlight unanswered questions in the area of XCI initiation inhuman development.

1Universite de Paris, UMR 1016, Institut Cochin, 75014 Paris, France. 2Service deBiologie de la Reproduction – CECOS, Paris Centre Hospital, APHP.centre, 75014Paris, France. 3Universite de Paris, Epigenetics and Cell Fate, CNRS, F-75013Paris, France.

*Authors for correspondence (catherine.patrat@aphp.fr;claire.rougeulle@univ-paris-diderot.fr)

C.P., 0000-0003-4885-8885; J.-F.O., 0000-0002-4191-6336; C.R., 0000-0003-3861-4940

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Dynamics of X chromosome activity during early humandevelopmentTo gain insight into the initial phases of human X chromosomeinactivation during early development, scientists rely on embryosderived from in vitro fertilization (IVF), which raises ethical as wellas technical issues (see Box 2). Below, we summarize our currentknowledge in this area of research.

Sex-dependent differences in early embryonic developmentAs the Y chromosome is enriched in transcription-regulating genes,which could accelerate growth development (Bellott et al., 2014), ithas been suggested that male and female embryos may differ inseveral parameters during the human pre-implantation period, as inother mammalian species (Kochhar et al., 2001). As thesedifferences appear before sexual differentiation, they cannot belinked to sex-related hormonal differences; instead, they could beattributed to a bias in the expression level of sex chromosome-encoded genes, which may indirectly affect the expression ofautosomal genes. In particular, several studies have reported that sexcould affect human embryo kinetics. The number of cells in day 2IVF-conceived male embryos is greater than that in female embryos

(Ray et al., 1995). This observation was later corroborated by thefinding that male embryos tend to reach the final blastocyst stagesfaster than females (Alfarawati et al., 2011; Hentemann et al., 2009;Luna et al., 2007; Ménézo et al., 1999). Because the faster-developing and most expanded blastocysts are often selected forembryo transfer, this could explainwhy the sex ratio at birth after IVFhas been reported to be skewed towards the male sex (Chang et al.,2009; Luna et al., 2007;Ménézo et al., 1999). Increasedmitotic ratesin male embryos could be attributed to early expression of sexdetermining region Y gene (SRY) from the Y chromosome, which isknown to have mitogenic properties and is expressed as early as theeight-cell stage in humans (Fiddler et al., 1995). These differences inproliferation kinetics, which only become apparent around the timeof embryo genome activation (EGA; see Glossary, Box 1), may alsobe explained by the dose-dependent transcription levels of mRNAs,notably from the X chromosome. Indeed, the differential expressionof genes between sexes during pre-implantation development hasrecently been confirmed (Zhou et al., 2019). The majority of earlydifferential gene expression (detected between day 3 and day 5) is X-linked, whereas at day 6 and day 7 it is mainly autosomal and mayresult from indirect effects of early X-linked gene expressionimbalance. Different proliferation rates of male and female embryosmay alternatively result from different metabolic activity betweenthe two sexes. Some X-linked genes are related to amino acidturnover and transport, including solute carrier family 38 member 5(SLC38A5), which transports asparagine, one of the amino acidsdifferentiallymetabolized in female andmale embryos (Picton et al.,2010); other X-linked genes, such as glucose-6-phosphatedehydrogenase (G6PD), are involved in the metabolism of glucose.

However, other studies have challenged the sex dependence of earlyhuman development as they did not confirm that male human pre-implantation embryos cleave at a faster rate (Csokmay et al., 2009;Richteret al., 2006), have a higher developmental potential (Ben-Yosefet al., 2012) or exhibit different kinetic parameters (Bronet et al., 2015;Serdarogullari et al., 2014) or inner cell mass and trophectoderm (TE)morphology (Alfarawati et al., 2011) compared with female embryos.Therefore, although the existence of sex-based difference in earlyembryonic development is an area of active debate, and thus requiresfurther investigation, there is some indication of the differentialexpression of X-linked genes between early male and female humanembryos, which raises the issue of dosage compensation.

X chromosome dosage compensationOur knowledge of X chromosome dynamics in early developmenthas mostly come from studies in mice, in which XCI is imprintedduring the pre-implantation window and in extra-embryonic tissuesthat give rise to the placenta (Takagi, 2003). However, it has beenknown for decades that sex chromosome aneuploidies in humanshave different consequences than those in mice. For example,mouse embryos with a supplementary copy of an X chromosome(XXX or XXY) die only when this supplementary chromosome isinherited from the mother (Xm) (Goto and Takagi, 1998). This hasbeen interpreted as a resistance of the Xm to early XCI, which isacquired during the growth phase of the oocyte and involves amaternal-specific H3K27me3 imprint at Xist (Inoue et al., 2017). Incontrast, humans with supernumerary X chromosomes survive anddevelop normally, even if the supplementary chromosome ismaternally inherited (Skakkebaek et al., 2014). In both XXXfemales or XXY men (Klinefelter syndrome), only one Xchromosome is active owing to XCI of all extra chromosomes(Tuttelmann and Gromoll, 2010), independent of the parentalorigin, thus arguing against imprinted XCI in humans. Taking

Box 1. GlossaryEmbryo genome activation (EGA). A complex process occurring in theembryo after fertilization. It allows the embryo to take control over itsdevelopment through a faithful reprogramming and restructuring of thetwo parental genomes before transcription occurs.ESHRE Istanbul consensus. Defines the morphological criteria toclassify a pre-implantation embryo at different stages of its development(Alpha Scientists in Reproductive Medicine and ESHRE Special InterestGroup of Embryology, 2011).Heteromorphic. A chromosome pair, such as the sex chromosomes Xand Y, which have some homology but differ in size, shape and geneticcontent.Hysterectomy. Surgical ablation of the uterus.Long noncoding RNA (lncRNA). An RNA molecule longer than 200nucleotides that does not encode a protein.Pluripotency. The property of some cells to form all somatic lineagesand germ cells; pluripotency exists in two states, naïve and primed,representing the distinct cellular features of the pre- and post-implantation epiblast, respectively.Primordial germ cells (PGC). Precursors or primary undifferentiatedstem cells that will give rise to the gametes.Random XCI (rXCI; see XCI). Refers to the equal probability of each Xchromosome (maternal and paternal) to be inactivated. Random XCIresults in a mosaic individual, in which the maternal X chromosome isactive in roughly half of the cells and the paternal one in the other half.RNA-seq. Next-generation sequencing of all cellular RNAs, which canbe performed on a cell population (bulk RNA-seq) or on individual cells(scRNA-seq).Topologically associated domains (TAD). Chromosome domains ofpreferential interaction in the 3D space.Xa. Active X chromosome.XACT (X-active coating transcript). An lncRNA that coats active Xchromosomes in early development. XACT is found only in hominoids.The function of XACT is unknown.XCI. X chromosome inactivation, the process whereby one of the two Xchromosomes is transcriptionally shut down in female mammals.Xe. An originally inactive X chromosome that has been partiallyreactivated owing to erosion of XCI, a process that takes placespontaneously when pluripotent stem cells are grown in culture.Xi. Inactive X chromosome, with most genes being silenced.XIST (X-inactive specific transcript). An lncRNA produced by the Xchromosome that is retained in the nucleus and triggers XCI. XIST isfound in all placental mammals.

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advantage of the large number of human X-linked single nucleotidepolymorphisms (SNPs), analysis of X chromosome expression hasshown that the term placenta is composed of relatively large clonalpopulations with either the paternal or the maternal X chromosomeinactivated, suggesting randomXCI in this organ (Moreira de Mello

et al., 2010). This may explain why earlier studies performed onsingle placenta isolates, which may be composed of a clonalpopulation of cells, concluded that XCI is completely skewed (Gotoet al., 1997; Harrison, 1989; Ropers et al., 1978). However, recentdata based on RNA sequencing and methylation analysis supports aslight bias towards inactivation of the paternal X chromosome in thehuman placenta (Hamada et al., 2016).

The analysis of pre-implantation embryos confirmed the lack ofimprinted XCI and provided striking findings regarding dosagecompensation in early human development. The initial observationthat XIST is expressed from the maternal X chromosome in pre-implantation embryos and blastocysts further confirmed lack ofpaternal-specific XCI in humans (Daniels et al., 1997; Ray et al.,1997). This finding was later supported by analyses at the single celllevel (Briggs et al., 2015; Okamoto et al., 2011; Petropoulos et al.,2016; Vallot et al., 2017). XIST expression starts from the four-cellstage (Fig. 1), coincident with the onset of EGA, and graduallyincreases in an asynchronous manner in individual blastomeres(Briggs et al., 2015). What is particularly striking is the observationthat XIST is expressed in male embryos and from both Xchromosomes in females (Okamoto et al., 2011; Petropouloset al., 2016; Vallot et al., 2017). In situ studies revealed bi-allelicXIST coating in the majority (∼85%) of cells of day 6 and day 7female blastocysts (Fig. 2; Okamoto et al., 2011; Petropoulos et al.,2016; Vallot et al., 2017). In male embryos, ∼60% of cells exhibitaccumulation of XIST RNA on the single X chromosome in day 5blastocysts and less than 10% at day 7 (Okamoto et al., 2011;Petropoulos et al., 2016). Notably, XIST expression is significantlyhigher in female than in male embryos (Moreira de Mello et al.,2017; Petropoulos et al., 2016).

Given the major contribution of Xist to XCI demonstrated in themouse, this unusual pattern of XIST expression immediately raisedquestions regarding the activity of the X chromosomes during thepre-implantation period in humans. Analysis of selectedX-chromosomal genes revealed bi-allelic expression in the morulaand blastocyst (days 5 and 6) and a lack of H3K27me3 enrichment,

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Fig. 1. Comparison of XCI timing and hallmarks betweenmouse and human early development.During female mouse pre-implantation development (top),X chromosome inactivation (XCI) is tightly coupled to Xist expression and occurs in two independent waves (blue and green, respectively). Imprinted XCI (iXCI),with systematic inactivation of the paternal X (Xp) is established rapidly in the mouse, following embryonic genome activation (EGA), and is maintained up to theformation of the early blastocysts, where iXCI is reversed in the cells of the inner cell mass (but maintained in the extra-embryonic lineage, not depicted). This isfollowed by random XCI (rXCI), in which either the maternal X (Xm) or the Xp is inactivated. Throughout mouse development, H3K27me3 patterns (purple) followXist coating on the X-chromosome (green). Human female pre-implantation development (bottom) unfolds without iXCI, despite expression of XIST upon EGAfrom both Xm and Xp. The X-linked XACT lncRNA is also expressed throughout pre-implantation development (red). H3K27me3 accumulation on the Xchromosome does not follow XIST expression but correlates with the repression of XACT. rXCI initiates at the peri-implantation stage, with kinetics that remain tobe determined (see text).

Box 2. Issues related to the use of human embryosIn contrast to mice, and with the exception of studies involving womenundergoing elective hysterectomy (see Glossary, Box 1; Hertig et al.,1956), all observations on human embryo development come fromembryos derived from in vitro fertilization (IVF). The evolution ofbioethical laws authorizing, in many countries, research on humanembryos to be performed under specific legal conditions has recentlypermitted increased research efforts into early human development. It is,however, important to remember that most IVF embryos have beenconceived in a context of parental infertility, using potentially abnormal orimmature gametes, and with assistance from several in vitro steps,including fertilization and the culturing of gametes and embryos. Humanembryos are also characterized by a high level of aneuploidy, most ofthem originating from meiotic errors in oocytes (5-20% for oocytes),which is higher than in other species (0.5-1% for murine germ cells), andincreaseswith maternal age (Nagaoka et al., 2012). All these parametersare likely to impact embryo quality per se. Another important issue is therelative inefficiency of human embryos to reach the blastocyst stage.Only up to 40-60% of cleaved embryos develop further into blastocysts invitro (Glujovsky et al., 2016), a number similar to the 50% in vivoestimates (Hertig et al., 1956), which reflects the low fecundity rates ofhumans. Therefore, only few human embryos exhibit typical kinetics ofdevelopment. Finally, in most basic research studies, embryos are notclassified according to international guidelines used routinely in IVFpractice, such as the ESHRE Istanbul consensus (see Glossary, Box 1;Alpha Scientists in Reproductive and ESHRE Special Interest Group ofEmbryology, 2011). They are rather described as ‘typical’ day-5 or day-6embryos rather than with strict kinetic-morphological criteria, whichoccasionally leads to difficulties in defining their exact developmentalstage. This imprecise morphological description may explain somediscrepancies between studies.

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despite the accumulation of XIST (Okamoto et al., 2011). Bi-allelicexpression of X-linked genes has since been confirmed at thechromosome-wide level and across pre-implantation developmentas early as EGA at day 3 (Zhou et al., 2019) and with the degree ofbi-allelic X chromosome expression remaining stable from day 4 today 7 (Petropoulos et al., 2016). When normalized to the totalamount of X chromosome transcripts per embryonic cell, females,but not males, show a decrease in X chromosomal gene expressionbetween the morula (day 4) and late blastocyst stages (day 7), whichwas interpreted as X chromosome dampening (Petropoulos et al.,2016). The model of X chromosome dampening was, however,questioned when different criteria for computational analysis of thedata were applied (Moreira de Mello et al., 2017).All of these observations indicate that human pre-implantation

development proceeds in the absence of complete XCI andcorresponds to a pre-inactive status (pre-XCI, Box 3 and Fig. 2),thus providing the first and only context known so far in which XISTaccumulation is not correlated to XCI. This highlights the need tomonitor multiple parameters and combine various approaches to

assess the XCI status (Box 3) and raises questions as to themechanisms and timing of XCI in humans.

Timing of XCI: when does it take place?Owing to limitations in accessing relevant biological material, onlyfew studies have so far addressed the timing of XCI. In an in vitromodel for human implantation, H3K27me3 foci, characteristic ofthe Xi in female somatic cells, were observed in ∼25% of TE cellsand in 7.5% primitive endoderm (PE) cells at day 8, but not inepiblast (EPI) cells (Teklenburg et al., 2012). It has also beenproposed, based on the presence of H3K27me3, that one Xchromosome is inactivated in 4-week somatic embryonic cells(Tang et al., 2015). XCI thus initiates between early implantationand the end of the first month of pregnancy, possibly in aprogressive manner and with different kinetics depending on the celllineage and its timing of specification. Because extended in vitroculture in humans has long been inefficient or not allowed forethical reasons, this period of human development and theassociated gene regulatory processes remained unexplored.

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Fig. 2. The various flavors of X-chromosome activity during human development and in cellular models. (A) In pre-implantation embryos,XIST and XACTlncRNAs are expressed following embryonic genome activation (EGA) and co-accumulate on active X chromosomes (Xa). At these stages,XIST coating appearsto be more diffuse than in differentiated cells. Following implantation, XACT expression is lost, XIST becomes mono-allelic in female cells and triggers XCI on onerandomly chosen X (Xi); this status is thought to be stably maintained and propagated in somatic cells. Reversal of XCI is observed in primordial germ cells(PGCs), which maintain a certain level of XIST that is possibly not tethered to the X chromosomes. (B) In vitro pluripotent cellular models include humanembryonic stem cells (hESCs) that can bemaintained in naïve and primed states of pluripotency, which bear resemblances to pre- and post-implantation stages ofin vivo development, respectively. In the naïve state, XIST and XACTmay co-accumulate on two active X chromosomes, with XIST being more dispersed than indifferentiated cells, similar to pre-implantation embryos. Of note, in naïve hESCs, XIST is often expressed from only one X chromosome. Primed hESCs are in apost-XCI state in which one chromosome is inactivated (Xi; coated by XIST and enriched in H3K9me3/H3K27me3 marks) and the other one is active (Xa; coatedby XACT). These primed hESCs spontaneously undergo erosion of XCI, in which expression and coating of XIST and enrichment of H3K27me3 marks, but notH3K9me3marks, are lost from the eroded X (Xe). Erosion of XCI initiates with coating of the Xe by XACT and leads to re-expression of a subset of genes from Xe.Differentiation of naïve cells triggers XCI on one chromosome, whereas differentiation of primed hESCs does not appear to alter their XCI status; althoughshuffling of epigenomic marks may occur (see text), eroded cells maintain their partially inactivated Xe (Mekhoubad et al., 2012; Vallot et al., 2015).

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However, the recent design of novel in vitro systems allowinghuman embryos to progress and organize beyond the blastocyststage (Deglincerti et al., 2016; Shahbazi et al., 2016) will be helpfulin lifting the veil on XCI initiation. Allele-specific expression of X-linked genes from day 6 to day 12 in vitro cultured embryos suggeststhat rXCI initiates during the implantation window, but is not fullycompleted by day 12 (Zhou et al., 2019). More studies will beneeded to ascertain these observations.

X chromosome reactivation in the germlineThe second round of whole-genome reprogramming occurs in thegermline. The X chromosome is particularly concerned as the Xi isreactivated in the female germline. Although germline Xchromosome reactivation (XCR) has been substantially studied inmice (Chuva de Sousa Lopes et al., 2008; de Napoles et al., 2007;Sugimoto and Abe, 2007), its timing in humans is still unclear.PGCs are the precursors of mature gametes – the sperm and

oocytes in mammals. Using global analysis of X chromosomeexpression and allelic investigation of selected genes, of which

some were known to escape XCI, it was initially concluded thatXCR has already taken place in the earliest female human PGCs(hPGCs) analyzed, at 4 or 5.5 weeks after fertilization (Guo et al.,2015; Li et al., 2017). Intriguingly however, the total expressionlevel of X-linked genes in female PGCs is less than twofold that ofmales between 4 and 11 weeks of development (Guo et al., 2015).This could indicate either a global increase of the single Xchromosome expression in male PGCs, a decrease in activity of thetwo X chromosomes in females (‘dampening’), or a combination ofboth. The conclusion that XCR has fully occurred in 4-week PGCshas later been challenged and a more heterogeneous pattern, with∼30% of hPGCs at 4-9 weeks still exhibiting incomplete XCR, wasreported (Vértesy et al., 2018). This is consistent with some of theseearly hPGCs displaying a faint but characteristic perinuclear spot ofH3K27me3, indicative of XCI (Vértesy et al., 2018).

Together, these results show that XCR in hPGCs is aheterogeneous and complex process, starting from 4 weeks ofdevelopment onward and occurring in an asynchronous manner.This correlates with the global wave of DNA demethylation, whichis already underway in 4-week PGCs (Guo et al., 2015; Tang et al.,2015). The onset of XCR appears to be related to the transcriptionalsignature of the cell rather than the fetal age (Vértesy et al., 2018).Importantly, XIST is expressed in the male and female humangermline independently of the XCR status and at all stages analyzed(Gkountela et al., 2015; Vértesy et al., 2018); XIST is also expressedin oocytes, albeit to a lower extent (Daniels et al., 1997; Ernst et al.,2018). It remains to be determined whether XIST accumulatesaround the X chromosomes in hPGCs but, in any case, suchwidespread XIST expression is not associated with H3K27me3enrichment on the X chromosomes (Tang et al., 2015). Altogether,these observations suggest that, similar to pre-implantationdevelopment, the activity status of the X chromosome in thefemale human germline does not depend on the presence of XIST,but rather on its ability to trigger chromosome silencing, althoughthe underlying mechanisms still remain elusive. The developmentof protocols to generate PGC-like cells from pluripotent stem cells(PSCs) (Irie et al., 2015; von Meyenn et al., 2016) opens newperspectives for studying XCR in the germline.

PSCs as a cellular model for investigating XCIEven though recent advances have been made regarding XCI inhuman development using pre-implantation embryos, the paucity ofsuch material and, more importantly, the associated ethical issuesprevent their extended use. PSCs, which possess the formidablecapacity of surviving quasi indefinitely in culture, provide apromising ex vivo counterpart to these early developmental stages,and thus stand as the ultimate cellular model for human XCI studies.Indeed, mouse PSCs have been instrumental for decipheringfeatures, mechanisms and regulators of XCI in rodents. Two maintypes of PSCs exist: embryonic stem cells (ESCs), which aredirectly derived from embryos, and induced pluripotent stem cells(iPSCs), which are obtained through in vitro reprogramming ofdifferentiated cells. Because the question of whetherreprogramming of differentiated cells to iPSCs is accompanied byXCR in human as it has been shown in the mouse (Maherali et al.,2007) is still debated (Talon et al., 2019), we have chosen to onlydiscuss ESCs.

Human ESCs in a pre-XCI statusAnalogous to mouse ESCs, initial reports on femaleundifferentiated human ESCs (hESCs) showed that they carry twoactive X chromosomes (Xa; see Glossary, Box 1) that do not express

Box 3. How to probe XCI status in human cellsPartial assessment of XCI status based on limited criteria may, and hasin the past, lead to inaccurate conclusions. Cells can either beconsidered pre-XCI [containing two active X chromosomes (XaXa)] orpost-XCI (once XCI has been initiated). Classical post-XCI cells carryone active X and one inactive X (XaXi). In hESCs however, XCI isunstable and thus post-XCI cells may harbor one active X and onepartially reactivated ‘eroded’ X (XaXe). The following experimentalstrategies are used to determine the XCI status in vivo and in vitro.X chromosome expressionThe most robust criterion to infer XCI status is to monitor thetranscriptional state of X chromosomal genes. Mono-allelic expressionof X-linked genes is indicative of a post-XCI state, whereas bi-allelicexpression suggests a pre-XCI state. However, 12-25% of humanX-linked genes escape XCI (Carrel and Willard, 2005), resulting in theirconstitutive or stage/tissues-specific expression, even from the Xi. Inaddition, allelic expression (mono- versus bi-) has to be assessed at thesingle cell level because XCI is, in theory, random. X chromosomeexpression can be assessed using RNA-FISH, which provides visualinformation for discrete X-linked genes, independently of the presence ofallelic SNPs. Using chromosome paint probes, RNA-FISH can also beused to monitor chromosome-wide expression, although the identity ofthe detected targets remains unknown (Vallot and Rougeulle, 2016).Alternatively, scRNA-seq simultaneously addresses the expression of allX-linked genes and, depending on the presence of informativepolymorphisms, provides allele-specific information regarding the XCIstatus.XIST expressionXIST expression can be monitored using RNA-seq or RNA-FISH, thelatter providing additional information regarding the distribution of theRNA at the single cell level (compaction of dispersed cloud versuspinpoint localization). However, in humans, expression andaccumulation of XIST is not, per se, indicative of XCI.Chromatin profilingH3K27me3marks the Xi in humans as it does in themouse. However, asfor XIST, presence or absence of this mark does not allow firmconclusions of pre- or post-XCI state. H3K9me3 appears to betterdiscriminate post- from pre-XCI cells (Vallot et al., 2017), but this requiresfurther investigation, notably in embryos. Histonemarks can bemonitoredusing immunofluorescence (IF) or chromatin immunoprecipitationfollowed by sequencing (ChIP-seq). DNA methylation of X-linked CpGislands is also a marker of human X-inactivation (Weber et al., 2007).

For routine analysis, RNA-FISH for selected X-linked genes,combined with XIST RNA-FISH and/or H3K27me3 IF is sufficient todistinguish between the various X chromosome states described so far.

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XIST (XaXIST−XaXIST−) (Dhara and Benvenisty, 2004). The activityof both X chromosomes is stabilized when cells are derived inhypoxia (Lengner et al., 2010), although the impact of oxygenconcentrations is under debate (Patel et al., 2017). However, itrapidly transpired that the X chromosome status of hESCs is farmore complex than initially thought, with various patterns foundbetween, and even co-existing within, hESC lines and cellpopulations (Hoffman et al., 2005; Shen et al., 2008; Silva et al.,2008; Vallot et al., 2015). In addition, the ability of XaXIST−XaXIST−

hESCs to undergo XCI upon differentiation is now questioned;instead, the X chromosome status of most undifferentiated hESCs ismaintained during differentiation (Patel et al., 2017). The reason forthis discrepancy is unknown, but may be linked both to the criteriaused to define the XCI status (Box 3) as well as to the heterogeneousnature of the starting population, with differentiation selecting onepopulation (XIST+) over another (XIST–). Nevertheless, studies inpre-implantation embryos have now established that the pre-XCI statein humans is XaXIST+XaXIST+ (Okamoto et al., 2011; Petropouloset al., 2016). This has launched the race to define hESC cultureconditions that sustain a pre-implantation-like state in vitro.Pluripotency (see Glossary, Box 1) exists in multiple ‘flavors’,

including naïve and primed (Davidson et al., 2015; Nichols andSmith, 2009), which correspond to distinct developmental stages,pre-implantation inner cell mass and post-implantation epiblast,respectively. These two states of pluripotency can be distinguishedby several metabolic and molecular features, such as theirdependency on signaling pathways and their transcriptionalsignatures, including those emanating from transposable elements(Theunissen et al., 2016). Epigenetic characteristics such as thelevels and distribution of DNA methylation also define naïve andprimed pluripotent states. The X chromosome activity status isanother hallmark – and as we will see, a robust one – of the variouspluripotent states, with naïve pluripotency being characterized by apre-XCI state and primed pluripotency by a post-XCI state (Fig. 2).Several methods and culture formulations have been defined tosustain naïve pluripotency in vitro, with variable outputs (reviewedby Collier and Rugg-Gunn, 2018). Confusion regarding Xchromosome activity in these different settings has arisen frompartial assessment of XCI and from using inappropriate criteria (i.e.XIST expression). In addition, some culture conditions aresuccessful in inducing several features of naïve pluripotency butnot in resetting the X chromosome status, suggesting that XCR is alate event in the process (Sahakyan et al., 2017b). There is thereforean intimate connection between XCI and naïve pluripotency:assessment of X chromosome activity is a powerful tool to define‘truly’ naïve cells and, conversely, naïve PSC are instrumental tostudy early stages of XCI. So far, two main culture formulationsreferred to as 5iLA (Theunissen et al., 2016, 2014) and t2iLGö(Takashima et al., 2014) are compatible with a pre-XCI status (Guoet al., 2017; Sahakyan et al., 2017b; Vallot et al., 2017), as definedby bi-allelic expression of X-linked genes. The accumulation ofXIST on active X chromosomes is another hallmark of pre-XCIstatus, with the pattern of XIST accumulation in those cells beingqualitatively similar to that of early embryos and more diffusecompared with post-XCI cells (Fig. 2; Sahakyan et al., 2017b;Vallot et al., 2017). However, the true equivalence of naïve hESCsto in vivo pre-implantation stages is questionable, notably as XIST ismostly expressed from only one, and rarely two X chromosomes(Sahakyan et al., 2017b; Vallot et al., 2017). Moreover, conflictingresults have been obtained regarding other hallmarks of XCI,notably H3K27me3. Although H3K27me3 was found to beenriched on active X chromosomes decorated by XIST in some

studies (Sahakyan et al., 2017b), others revealed a lack ofaccumulation of heterochromatin marks (H3K27me3 andH3K9me3) on XIST-expressing Xa in naïve cells (Vallot et al.,2017), similar to embryos (Okamoto et al., 2011). Therefore, furtheranalysis is required to fully assess the chromatin landscape of XIST-coated active X chromosomes in humans.

The fact that naïve hESCs capture, to some extent, the pre-XCIstatus offers, in theory, a unique opportunity to assess the initiationof human XCI, which has so far remained elusive. A directtransition from XaXIST+XaXIST− to XiXIST+XaXIST− has beenreported (Guo et al., 2017), whereas another study described anintermediate XaXIST−XaXIST− stage (Sahakyan et al., 2017b), whichis analogous to the initial observation of XaXIST−XaXIST− hESCs. Itremains to be determined whether the latter exists in developingembryos. Moreover, differentiation of naïve cells obtained frompost-XCI cells results in biased XCI, with the original Xi alwaysinactivated (Sahakyan et al., 2017b). This indicates that a memory ofthe previous inactivation status is left, again questioning the truenaivety of these cells. This issue might not apply to blastocyst-derived naïve hESCs, which are essentially deprived of XCImemory, and in which XaXIST+XaXIST+ might be stabilized moreefficiently (Sahakyan et al., 2017b).

What can we learn from primed hESCs?Although a cellular model that confidently mimics XCIestablishment is still lacking, primed hESCs, in which XCI hasalready occurred, might still be informative to understand the earlystages of XCI. Several pieces of evidence suggest that the post-XCIstatus of primed hESCs is distinct from that of differentiated cells.The most obvious feature is the instability of the inactive state inprimed hESCs, with several hallmarks of XCI being spontaneouslylost upon passages, a phenomenon that has never been reported inany other differentiated cells in culture, potentially with theexception of some, but not all, cancer cells (Bar et al., 2019;Chaligné et al., 2015). This erosion of XCI is characterized bydisappearance of XIST expression and partial reactivation of the Xi(Fig. 2; Mekhoubad et al., 2012; Vallot et al., 2015). The Xchromosome that underwent erosion is referred to as eroded X (Xe;see Glossary, Box 1). It appears that not all genes are similarlysusceptible to XCI erosion and, in the absence of XIST, genesilencing might be more efficiently maintained in some regionscompared with others (Bar et al., 2019; Patel et al., 2017; Vallotet al., 2015). The pattern that emerges from independent analyses isthat of a core domain flanking the centromere being resistant to XCIerosion, whereas the middle parts of both short and longchromosome arms are more prone to XCI instability (Bar et al.,2019; Patel et al., 2017; Vallot et al., 2015). The reason for this isunclear, but it might be linked to the organization of the Xichromatin into distinct territories. There is indeed a correlationbetween susceptibility to erosion and the pattern of histone marks,with regions normally enriched in H3K27me3 being preferentiallyreactivated over H3K9me3-marked regions (Vallot et al., 2015).This is probably linked to the fact that H3K27me3 is dependent onXIST and lost from the Xi in eroded cells, whereas H3K9me3 ismaintained (Vallot et al., 2015). In addition, erosion of XCI ischaracterized by partial promoter CpG demethylation (Nazor et al.,2012; Patel et al., 2017; Shen et al., 2008). Of note, the degree ofXCI instability might depend on the culture conditions (personalobservations, J-F.O. and C.R.). In summary, although XCI erosionis a culture artefact with seemingly no equivalence and/or relevancein normal development (Bar et al., 2019), it likely reflects peculiarand stage-specific Xi features. In agreement with this hypothesis,

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the chromatin organization of the Xi was found to distinguishpluripotent from differentiated cells (Vallot et al., 2015, 2016). Inpost-XCI hESCs, H3K9me3 and H3K27me3 chromatin marks aremutually exclusive and anti-correlated (Vallot et al., 2015).Intriguingly, such bimodal organization of the Xi is also observedin immortalized cells on both metaphase (Chadwick and Willard,2004) and interphase chromosomes (Chadwick, 2007; Nozawaet al., 2013; Vallot et al., 2016). In contrast, there is significantoverlap of H3K9me3 and H3K27me3 in primary differentiated cells(Vallot et al., 2015), thus enhancing XCI stability in a synergisticmanner. This is in agreement with previous studies that have shownthe redundancy of the multiple layers of epigenetic modificationsthat ensure stable maintenance of XCI (Csankovszki et al., 2001).Even if a potential impact of cell culture in remodeling the

epigenomic landscape of the X chromosome cannot be excluded,these observations may reflect a multistep establishment of the Xichromatin landscape. Future studies should also address thepresence of additional histone marks and variants that arenormally enriched on the Xi, such as H4K20me1, H2AK119Uband macroH2A.

Molecular control of human XCIContribution of XIST to human XCIXist has been established as the trigger of XCI in mice (Penny et al.,1996). Although it is implicitly acknowledged that XIST exertssimilar functions in all species, functional proof for this hypothesisis limited. Initial attempts to understand the function of human XISTrelied on the introduction of XIST genomic and inducible cDNAtransgenes on autosomes into various cell types of different origin:mouse ESCs, human adult male cancer cell lines and on one of thethree chromosomes 21 in iPSCs derived from Down syndromepatients. In all cases, the ectopic XIST RNA is able to spread and toinduce some level of silencing in cis, albeit to a different extent(Chow et al., 2007; Hall et al., 2002; Heard et al., 1999; Kelsey et al.,2015). In transgenic mESCs, silencing of autosomal genes occursonly upon differentiation, and only in a fraction of cells in cell lineswith high transgene copy numbers, even ifXIST is already expressedin undifferentiated cells (Heard et al., 1999). Autosomal integrationof a XIST genomic or cDNA transgene in human fibrosarcoma cellsalso leads to silencing of a nearby introduced reporter, and to globalchromatin and transcriptional reorganization of the transgenicchromosome (Chow et al., 2007; Hall et al., 2002), in a manner thatdepends on the integration site (Kelsey et al., 2015). XIST cDNAtransgene induction in Down syndrome cells efficiently triggerssilencing of the entire supernumerary chromosome 21, at least whenXIST is induced in a pluripotent context, leading to an almostnormal disomic level of expression for genes on chromosome 21(Jiang et al., 2013). More recently, loss-of-function approaches havebeen undertaken, in which thewhole XIST sequence or parts of XISThave been deleted in post-XCI cells, such as female embryonic orcancer cells (Lee et al., 2019; Lv et al., 2016). All the deletions thatresult in loss of XIST expression lead to some degree of XCR,questioning the idea that XIST is dispensable for XCI maintenance(Brown and Willard, 1994). Taken together, these pieces ofevidence suggest that human XIST is central to XCI, although theultimate demonstration of XIST function in a more physiologicallyrelevant context, such as naïve hESC differentiation, whichrecapitulates the initiation of XCI, is still lacking.Analysis of XCI in hESCs has also pointed to the existence of

XIST-independent silencing events; signs of X chromosome re-inactivation have been detected upon differentiation of erodedhESCs, in the absence of XIST re-expression. For example, although

hESCs with eroded XCI display two X chromosome transcriptionterritories, only one persists upon differentiation, and this isaccompanied by re-silencing of individual X-linked genes (Vallotet al., 2015). Analysis of differentiated derivatives of hPSCs alsoshowed that monoallelic expression of the X chromosome isexpanded during differentiation, independently of XIST (Bar andBenvenisty, 2019). In all of the above cases, re-silencing of the Xe isonly partial (Bar and Benvenisty, 2019; Mekhoubad et al., 2012;Patel et al., 2017; Vallot et al., 2015). Although the exactmechanisms of re-silencing are still elusive, the previousaccumulation of XIST appears to have left a memory of theinactive state, likely at the chromatin level, that is not sufficient tomaintain silencing of some X-linked regions in pluripotent cells, butwhich could be re-established upon differentiation. It is possible, forexample, that some chromatin marks such as H3K9me3, which areleft intact on the Xe in eroded cells but confined to specific domainsof the X chromosome, would spread to flanking regions in the 2D or3D space upon pluripotency exit and facilitate local repression.Assessment of the chromatin organization of the Xe in differentiatedcells should help resolve this issue.

Uncoupling XIST from XCIHuman early embryogenesis, as studied in naïve hPSCs, provides anunprecedented context in which XIST expression is uncoupled fromregular XCI. This implies that, in contrast to mice, in which Xistaccumulation systematically triggers XCI, XIST silencing activity istightly regulated in humans. The mechanisms involved are currentlyunknown, but several, non-mutually exclusive hypotheses can beproposed (Fig. 3).

First of all, it is still unclear whether XIST, despite accumulatingto some extent in the nucleus of pre-implantation embryos and naïvehESCs, is truly recruited to X chromosomes. Indeed, the pattern ofXIST accumulation is more dispersed in these contexts comparedwith post-XCI cells (Sahakyan et al., 2017a; Vallot et al., 2017),which is reminiscent of observations in mouse/human cell hybridsin which XIST spreads out on an active human X chromosome(Clemson et al., 1998). However, whether this is because of loosecontact with the X chromosome or normal interaction with a lesscompacted active X chromosome is to be determined. InvestigatingXIST distribution relative to the X chromosome territories shouldhelp solve this issue.

Another possible explanation for X chromosome activity in thepresence of XIST involves the absence of XIST effectors, i.e. factorsinteracting with XIST and mediating its silencing activity. Candidateand unbiased biochemical RNA-centric approaches, as well asgenetic screens, have, over the years and mostly in mice, led to theidentification of a myriad of factors that interact directly with Xistand/or contribute to the capacity of Xist to induce XCI (Chu et al.,2015; McHugh et al., 2015; Minajigi et al., 2015; Moindrot et al.,2015; Monfort et al., 2015). Among the latter are transcriptionalco-repressors (e.g. SPEN), nuclear matrix or compartmentalizationproteins (e.g. LBR and HNRNPU) and members of RNAmodification complexes (e.g. RBM15 and WTAP), which targetXIST RNA itself; however, this list is far from complete. Other XISTpartners, such as HNRNPU, are important for XistRNA localizationand association with the X chromosome territory (Hasegawa et al.,2010; Yamada et al., 2015). All these factors contact Xist on more orless specific subdomains of the RNA, notably key repeat regionssuch as the A-repeat, which is indispensable for Xist silencingactivity (Wutz et al., 2002). Of course, it remains to be determinedwhether the functions of the murine interactors of Xist in theX-inactivation process are conserved in humans. To allow

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X-chromosome activity in the presence of XIST, these XCI effectorsmay be transiently non-functional, or unable to localize to the Xchromosome, possibly because of impaired interaction with XIST.This could be owing to specific patterns of post-translationalmodification of XIST partners, post-transcriptional modification ofXIST RNA or to alternative splicing events that would splice outcrucial interacting regions of XIST. In this context, it is of interestto emphasize the lack of H3K27me3 enrichment on the Xchromosome in early embryos, as XIST accumulation andH3K27me3 enrichment have so far been entirely correlated.In a second scenario, as yet unknown factors might prevent XIST

from silencing the X chromosome. An interesting candidate is thelncRNA X-active coating transcript (XACT; see Glossary, Box 1).

XACT is an X-linked transcription unit of more than 250 kb, located50 Mb distal from the XIST gene (Fig. 2), which was discovered inhumans and is conserved in hominoids, but not in lower apes or inmore distantly related species (Vallot et al., 2013). The most strikingfeature of XACT is the nuclear distribution of the XACTRNA, whichdecorates active X chromosomes (Vallot et al., 2013). XACT wasoriginally identified in primed PSCs, in which XACT enrichment onthe Xa mirrors the accumulation of XIST on the Xi (Vallot et al.,2013). In embryos, XACT expression starts from the four-cell andthe eight-cell stage, concomitantly to XIST (Petropoulos et al., 2016;Vallot et al., 2017) (Figs 1 and 2). It accumulates on both active Xchromosomes, together with XIST, until the late blastocyst stage(expanded hatching blastocyst), where its expression variesaccording to the cell fate: XACT expression is maintained in EPIand PE cells, but tends to be shut down in future TE cells, whereasXIST remains expressed. XACT is also expressed from both Xchromosomes together with XIST in naïve hESCs (Fig. 2). In bothcontexts, XACT and XIST occupy distinct spatial domains and XACTcould hamper efficient XIST spreading and accumulation along theX chromosome, in agreement with its dispersed pattern.Alternatively, XACT could prevent the interaction between XISTand its effectors.

Regulation of XIST expression: the human XICThe distinct dynamics of XIST expression during earlyembryogenesis in humans and mice suggest that its regulatoryapparatus has diverged substantially across evolution. However, thestudy of chromosomal rearrangements in both species pointed to amajor contribution of the XIC, a domain of the X chromosomeencompassing XIST and multiple noncoding genes (Fig. 4) (Auguiet al., 2011). Several of these elements have been functionallyimplicated in the XCI process in mice, mostly through the regulationof Xist expression. The mouse Xic is physically and spatiallyorganized in two topologically associated domains (TADs; seeGlossary, Box 1) (Nora et al., 2012), one that contains elements thatfavor XCI, including Xist, and the other containing repressors of Xistand/or XCI (Fig. 4). The fact that the XIST ON state appears as thedefault one (Petropoulos et al., 2016; Vallot et al., 2017) raisesquestions as to the functional conservation of the repressors, at leastin the early stages of embryonic development. Among theserepressors is TSIX, the XIST antisense RNA. Tsix transcription inmice spans across the entire Xist gene and prevents Xist upregulationin cis (Lee and Lu, 1999). Tsix acts, at least in part, by triggeringchromatin remodeling across the Xist promoter region, in a mannerthat depends on Tsix transcription, but not on Tsix RNA (Navarroet al., 2006, 2005; Sado et al., 2005). A Tsix ortholog has beendescribed in humans, but similarities to the mouse counterpart interms of sequence and expression patterns are limited (Migeon et al.,2001, 2002), suggesting that it does not participate in XISTregulation. The lack of a Tsix equivalent in humans is likely to belinked to the absence of imprinted XCI in this species.

Among the Xic-linked Xist activators that have been characterizedin mice are the two noncoding genes Jpx and Ftx, which lie 5 and150 kb, respectively, upstream of Xist, within the Xist TAD (Fig. 4).Although both contribute to promoting Xist accumulation, they doso through distinct mechanisms. Jpx RNA controls Xist expressionin trans (Sun et al., 2013; Tian et al., 2010), whereas Ftx acts in atranscription-dependent manner to promote Xist expression in cis(Furlan et al., 2018). In contrast to Jpx, Ftx RNA is mainlydispensable for XCI initiation (Furlan et al., 2018). Both Jpx and Ftxhave orthologs in humans, and are part of the same TAD as XIST, buttheir role has remained elusive. Single cell RNA sequencing

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Fig. 3. Different scenarios for uncoupling XIST accumulation from XCIduring early human development. During pre-implantation humanembryonic development, XIST coating of X chromosomes does not triggerXCI. Multiple non-exclusive scenarios could explain this human-specificuncoupling. Induction of XCI could be prevented because of improper XISTlocalization/tethering to the X-chromosome (i), whichmayormay not beXACT-dependent. XIST activity could also be prevented by the action of XACTeffectors, which could antagonize XIST effectors (ii). Alternatively, XISTeffectors may not be expressed/active during these stages of development (iii).Finally, XIST might not be able to recruit its effectors because of defectiveprocessing such as mis-splicing or editing (iv). Whatever the scenario at stakein pre-implantation embryos, XIST coating eventually triggers X-chromosomesilencing following implantation.

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(scRNA-seq; see Glossary, Box 1) datasets from human pre-implantation embryos revealed that, within the XIC, JPX is theearliest gene to be activated, before XIST (Rosspopoff et al., 2019preprint). Functional investigation in naïve and primed hESCsfurther showed a major contribution for JPX in XIST expression

(Rosspopoff et al., 2019 preprint). However, unlike its mousecounterpart, it is JPX transcription, and not RNA, that regulatesXIST expression in humans (Rosspopoff et al., 2019 preprint). Thispoints to an unanticipated functional plasticity of orthologouslncRNA genes involved in XCI.

Cdx4

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Fig. 4. X-inactivation center in mouse and human. The X-inactivation centers (Xic) of mice (top) and humans (bottom) are organized in two conservedtopologically associated domains (TAD) that segregate the cis-interacting DNA elements within the XIC. In this representation, the intensity of the red colorcorrelates with the frequency of the interaction between the connected DNA elements. Positive and negative regulators of XCI are segregated on the chromosomeand in space through constrained interactions within the XIST and TSIX TADs, respectively. Although the overall organization (linear and 3D) is conservedbetween mouse and human, it is notable that fewer interactions are detected within the human TSIX TAD compared with the mouse Tsix TAD. This observationcould reflect the absence of a TSIX repressive regulatory function on XIST expression in humans (see text).

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Altogether, it appears that, although XCI in humans is ascrucially dependent on XIST as it is in mice (and likely in alleutherians), the XIST regulatory network might have divergedsubstantially during evolution. There are, however, many questionsthat remain to be addressed regarding the contribution of conservedand species-specific elements, notably noncoding genes, to XCI inhumans.

Conclusion and future perspectivesFundamental research on human embryos has only begun, yet it hasalready revealed unanticipated differences in XCI compared withthe mouse. There are, however, still many more questions unsolvedthan answers provided, in particular regarding the timing of XCIinitiation and reversion, the precise sequence of molecular events,the elements that control the capacity of XIST to induce silencing,and the XIST regulatory network. There is no doubt that acombination of models and approaches will be required toprogress our understanding of this key epigenetic regulatoryprocess. Mimicking embryo implantation by co-culture orexpanding the repertoire of models, for example to study embryodevelopment in a 3D environment, would undeniably be a majorasset. In addition, research on XCI will benefit from theimprovement of culture conditions that derive and maintainkaryotypically stable and truly naïve hESCs. This will allowresearchers to decipher the onset of X chromosome silencing, withthe important parameter that in cellulo studies need, as much aspossible, to be validated in embryos. Deciphering common themesand species-specificity of human XCI will impact a plethora ofresearch fields including human embryonic development, stem cellbiology, lncRNAs and X-linked diseases. Finally, on a more generalnote, the study of human XCI will contribute to our knowledge ofthe plasticity of regulatory networks and mechanisms in evolution, akey aspect to understand the long-sought articulation betweengenotype and phenotype.

AcknowledgementsWe are grateful to Edith Heard for helpful discussions and apologize to colleagueswhose work could not be cited owing to space constraints.

Competing interestsThe authors declare no competing or financial interests.

FundingC.P. is supported by a Qlife grant (Universite de Recherche Paris Sciences etLettres) and the Human Developmental Cell Atlas program (Institut National de laSante et de la Recherche Medicale). J.-F.O. and C.R. are supported by the EU‘EpiGeneSys’ Network of Excellence (FP7 Health, HEALTH-F4-2010-257082), theAgence Nationale de la Recherche (ANR-14-CE10-0017) and from the Ligue Contrele Cancer, the LabEx “WhoAm I?” (ANR-11-LABX-0071) and theUniversite de ParisIdEx (ANR-18-IDEX-0001) funded by the French Government through its’Investments for the Future’ program.

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